Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Effects of some thermal, chemical and mechanical treatments on lipase activity in Shammi goat milk

The effects of heating (20, 37 or 50 °C), cooling (5 °C), pasteurisation (71 °C for 15 s), boiling (100 °C), agitation (5 or 10 min), pH (acid or alkaline), and addition of chemicals such as silver and lead nitrates, copper sulphate and sodium chloride on lipase activity in Shammi goat milk were studied. There were non-significant differences (P < 0.01) in chemical composition between Shammi goat milk and Arabi cow milk. Lipase activity in Shammi goat milk was non-significantly (P < 0.01) lower than in Arabi cow milk. Lipase activity in milk of Shammi goats and Arabi cows was reduced when the milk was subjected to heating, cooling, pasteurisation, boiling, or when chemicals or acid was added, whereas in agitated and alkaline milk, the lipase activity was increased. The increase following agitation was greater after 10 min than 5 min. It can be concluded that heating, pasteurising, boiling, cooling, addition of certain chemicals and acidity are means by which lipase activity in milk can be reduced.     

Characteristics of lactococci cultures produced in commercial media

Strains ofLactococcus lactis ssplactis andL. lactis sspcremoris were propagated on milk, three commercial highly buffered media (HB media), and four commercial media designed for external pH control (EC media). With milk and HB media, fermentation was allowed to proceed until a pH of 4.9 was reached. With EC media, pH was maintained at 6.0 with 5 N NH₄OH. The cultures were analyzed for chain length, viable population, specific acidifying activity (SAA) and specific proteolytic activity (SPA). The starters were stored at 4° C for 3 days, and analyses for chain length, viable population and SAA were repeated. It was more difficult to standardize medium composition with the rehydrated commercial blends, as their titratable acidities had greater proportional variations than milk. As a rule, chain length was longer in fresh cultures than in the stored starters, andL. lactis sppcremoris cultures had longer chains thanL. lactis ssplactis. All commercial media produced starters with total populations at least as high as that obtained in milk. With the EC media, populations could be five times greater than with milk; increases were less important in HB media. The increase in population in EC and HB media was more marked withL. lactis ssplactis than forL. lactis sspcremoris strains. Storage at 4° C for 3 days did not significantly reduceL. lactis populations, but mortality (up to 70%) was observed withL. lactis sspcremoris. The overall SAA ofL. lactis ssplactis cultures in EC media was 35% lower than milk- or HB media-grown starters, but the greater populations reached in EC media enabled a significant reduction in inoculation rate. Some statistically significant correlations were obtained between SAA and SPA (positive) as well as with chain length (negative), but the coefficients of determination were generally very low. The drop in pH during storage at 4° C was less with HB media than in milk, and was in relation to their buffering capacity.     

FLIGHT ACTIVITY AND FATTY ACID UTILIZATION IN MYTHIMNA SEPARATA (WALKER) MOTHS*

Abstract The fatty acid (FA) compositions for total lipids from fat body, hemolymph and flight muscle of the armyworm moths, Mythirnna separata, at rest and after tethered flight for 1 h were determined by GC and GC-MS. The composition in these tissues comprises myristic acid (1%-2%), palmitic acid (more than 35%1, palmitoleic acid (9%-11%), stearic acid (less than 1%), oleic acid (about 32%), linoleic acid (12%-17%) and linolenic acid (3%-6%). After flight, FA level in the fat body, compared to that at rest, shows a significant decline at about 20 μg/mg tissue.h^-1; the concentration of FAs in hemolymph rises evidently, but change of FA content in flight muscle appears to be small. From the changes of proportional composition of FAs in fat body, hemolymph and flight muscle, it is found that the FAs selectively utilized for flight in flight muscle are predominantly the palmitic acid and oleic acid.     

Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter

Organic matter composition (lignin, holocellulose, 50% (v/v) methanol extract, water-soluble carbohydrate (WSC) and phenolics (WSP), petroleum ether extract, and ash) of A₀ layer soil treated with 700 g/m² of urea to promote ammonia fungi was investigated in a Japanese red pine (Pinus densiflora) forest. Nine species of fungi were found during the study period of 18 months after the treatment. Of these, seven species belong to the ammonia fungi. WSC content of the treated soil was lower than that of the control. Methanol extract content increased initially after the treatment, then decreased to below the control level. There were no consistent differences in other components between the treated plot and the control. The abilities to decompose cellulose, lignin, chitin, protein and lipid in 18 strains in 10 species of the ammonia fungi were also screened. Cellulose was not lysed byPseudombrophila deerata, Hebeloma spp. andLaccaria bicolor. Strong lignolytic activity was shown byLyophyllum tylicolor, Coprinus echinosporus andP. deerata. Chitin was decomposed byAmblyosporium botrytis, L. tylicolor, C. echinosporus andHebeloma vinosophyllum. All strains possessed proteolytic and lipolytic activities. Supply of glucose to the culture media resulted in weaker enzyme activities except for lignolytic ability.     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

PKA controls a level of topoisomerase I mRNA in mouse L5178Y lymphoma cells treated with db-cAMP

The level of topoisomerase I mRNA was measured in cells of two mouse lymphoma (LY) sublines treated with db-cAMP. A transient increase of the level was observed to be of about 60% of the basic level and to have maximum after the 3 h treatment of LY-S cells. The increase in LY-R subline was two-fold lower. The activity of PKA in a cytosol fraction of LY-S cells was 1.75 times higher than that in LY-R cells. The activity of PKA in membranes and nuclear fraction did not differ significantly in both cell types. When the activity of PKA in LY-S cells was inhibited with H8, no increase of the level of topoisomerase I mRNA was observed upon db-cAMP treatment of cells. We suggest that the activity of PKA in the cytosol controls the expression of topoisomerase I gene in LY cells at high concentration of cAMP.Abbreviations db-cAMP dibutyryl-cAMP - H8 N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide - LY mouse L5178Y lymphoma - PKA protein kinase A - topo I topoisomerase I     

Differential Requirements of Sodium for Coupling of Cannabinoid Receptors to Adenylyl Cyclase in Rat Brain Membranes

Abstract: Sodium is generally required for optimal inhibition of adenylyl cyclase by G_l/o-coupled receptors. Canna-binoids bind to specific receptors that act like other members of the G_l/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCI ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABA_B receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCI providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low K_m GTPase activity was maximal at 50 mM NaCI in striatal membranes, but was maximal in the absence of NaCI in cerebellar membranes. Assay of [³H]WIN 55212–2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other G_l/o-coupled receptors, because both NaCI and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions.